Plasmodium falciparum calcium-dependent protein kinase phosphorylates proteins of the host erythrocytic membrane

Abstract

The unusual Ca 2+-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62000, is activated by Ca 2+ ( K 0.5 = 15 μM) in the presence of Mg 2+ or Mn 2+, and can associate with 45Ca 2+. The activation by Ca 2+ could be partially replaced by Mn 2+, but not by Zn 2+ or Mg 2+. PfCPK preferentially phosphorylated casein and histone H1. The K m and V max for Mg 2+ ATP were 26 μM and 70 nmol min −1 mg −1, respectively, with casein as substrate; and 34 μM and 143 nmol min −1 mg −1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.